8 results on '"Daigeler, Anna‐Lena"'
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2. Intrazelluläres Trafficking des nierenspezifischen Natrium-Kalium-2Chlorid- Kotransporters
- Author
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Daigeler, Anna-Lena
- Subjects
NKCC2 ,TAL ,urogenital system ,thick ascending limb ,endocytosis ,600 Technik, Medizin, angewandte Wissenschaften::610 Medizin und Gesundheit ,Clathrin - Abstract
Der Na+-K+-2Cl--Kotransporter (NKCC2) der dicken aufsteigenden Henle-Schleife (thick ascending limb; TAL) ist essentiell für die Harnkonzentrierung und den Elektrolythaushalt. Das antidiuretische Hormon ADH aktiviert den Transporter über Stimulation seiner luminalen Translokation und Phosphorylierung an konservierten N-terminalen Threoninresten (T96, T101, and T114). Währenddessen wird NKCC2 in Lipid rafts eingeschlossen. Eine Deaktivierung des Transporters erfolgt über seine Dephosphorylierung und Internalisierung. Hierbei fungiert die Clathrin-abhängige Endozytose als eine der Hauptrouten. In der vorliegenden Arbeit wurde der Hypothese nachgegangen, dass die aktivierende, N-terminale Phosphorylierung von NKCC2 über Hemmung seiner Clathrin-abhängigen Internalisierung den Transporter in der Plasmamembran stabilisieren könnte. Eine Charakterisierung der Verteilung von NKCC2 in der apikalen Membran von TAL-Zellen zeigte eine anteilige Ko-Lokalisaton mit Clathrin in Non-raft Regionen, während der phosphorylierte Transporter sich vorwiegend in durch Flotillin-1 markierten Lipid rafts befand und kaum mit Clathrin ko-lokalisiert war. Eine pharmakologische Inhibition der Clathrin-abhängigen Endozytose mittels Chlorpromazin in kultivierten TAL-Zellen führte zur signifikanten Steigerung der NKCC2-Oberflächenexpression und untermauerte somit die Rolle dieser Internalisierungsroute im Kontext der NKCC2-Funktion. Die Untersuchung von N-terminalen phospho- sowie dephospho-NKCC2 Mutanten mittels Ko- Immunpräzipitation und GST-pull-down Assays deuteten darauf hin, dass die Phosphorylierung des Transporters seine N-terminale Bindung mit Clathrin inhibiert. Weitere Evidenz dafür wurde durch Stimulation von ADH-defizienten Brattleboro-Ratten mit dem ADH-Agonist dDAVP erbracht: die durch dDAVP induzierte Phosphorylierung von NKCC2 ging mit einer Abschwächung seiner Assoziation mit Clathrin einher. Zusammenfassend weisen die Ergebnisse der vorliegenden Arbeit darauf hin, dass die N-terminale Phosphorylierung von NKCC2 seine Clathrin-abhängige Internalisierung und somit seine Oberflächenexpression und Funktion moduliert., The Na+-K+-2Cl--cotransporter (NKCC2) of the thick ascending limb (TAL) plays an essential role in the urinary concentration and renal salt handling. Vasopressin stimulates NKCC2 activity by inducing its apical trafficking and phosphorylation at functionally relevant N-terminal residues (T96, T101, and T114 of rat NKCC2). The phosphorylated and thereby activated transporter is chiefly distributed in lipid raft membrane microdomains, whereas its deactivation is associated with dephosphorylation and clathrin-mediated internalization. Whether NKCC2 phosphorylation state affects its trafficking or surface expression was unclear. Therefore, the aim of this study was to evaluate effects of NKCC2 phosphorylation on its clathrin-mediated endocytosis. Evaluation of the topographic NKCC2 distribution in the apical membrane of TAL cells using antibodies that regnognize native NKCC2 or its phosphorylated form showed substantial co-localization of both signals with Lipid rafts markers such as flotillin-1 and a partial co-localization of native NKCC2 with clathrin in Non-raft membrane regions was detected. Pharmacological inhibition of clathrin-mediated endocytosis with Chlorpromazin in cultured TAL cells rapidly increased NKCC2 surface expression, suggesting an essential role of this internalization route for the transporters’ membrane turnover and function. Co-immunoprecipitation and GST Pulldown assays using N-terminal NKCC2 mutants mimicking its constitutive (de)phosphorylation, suggested that the N-terminal phosphorylation of the transporter may attenuate its association with clathrin-coated pits. Stimulation of NKCC2 phosphorylation in vivo by treating AVP-deficient Brattleboro rats with the V2 receptor agonist, dDAVP, reduced the interaction of the transporter with clathrin and increased its surface expression, thus providing further evidence for an interference between NKCC2 phosphorylation and its internalization rate. In summary, the results of this study suggest that N-terminal phosphorylation of NKCC2 attenuates its clathrin-mediated internalization and facilitates therefore its function by stabilizing the transporter in the luminal membrane.
- Published
- 2016
3. NKCC2 phosphorylation modulates its clathrin‐mediated internalization (892.20)
- Author
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Mutig, Kerim, primary, Daigeler, Anna‐Lena, additional, Dathe, Christin, additional, Wilson, Bridget, additional, and Bachmann, Sebastian, additional
- Published
- 2014
- Full Text
- View/download PDF
4. Annexin A2 mediates apical trafficking of renal Na‐K‐2Cl cotransporter (892.22)
- Author
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Dathe, Christin, primary, Daigeler, Anna‐Lena, additional, Jankowski, Vera, additional, Jankowski, Joachim, additional, Mutig, Kerim, additional, Bachmann, Sebastian, additional, and Paliege, Alexander, additional
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- 2014
- Full Text
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5. Annexin A2 Mediates Apical Trafficking of Renal Na+-K+-2Cl− Cotransporter
- Author
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Dathe, Christin, primary, Daigeler, Anna-Lena, additional, Seifert, Wenke, additional, Jankowski, Vera, additional, Mrowka, Ralf, additional, Kalis, Ronny, additional, Wanker, Erich, additional, Mutig, Kerim, additional, Bachmann, Sebastian, additional, and Paliege, Alexander, additional
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- 2014
- Full Text
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6. Pocket-size hand-held cardiac ultrasound as an adjunct to clinical examination in the hands of medical students and junior doctors
- Author
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Panoulas, Vasileios F., primary, Daigeler, Anna-Lena, additional, Malaweera, Anura S.N., additional, Lota, Amrit S., additional, Baskaran, Dinnish, additional, Rahman, Syed, additional, and Nihoyannopoulos, Petros, additional
- Published
- 2012
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7. Annexin A2 Mediates Apical Trafficking of Renal Na+-K+-2Cl- Cotransporter.
- Author
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Dathe, Christin, Daigeler, Anna-Lena, Seifert, Wenke, Jankowski, Vera, Mrowka, Ralf, Kalis, Ronny, Wanker, Erich, Mutig, Kerim, Bachmann, Sebastian, and Paliege, Alexander
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ANNEXINS , *BIOLOGICAL transport , *SODIUM ions , *PHOSPHORYLATION , *CELL membranes , *FUROSEMIDE - Abstract
Background: Regulated membrane expression of the renal furosemide-sensitive Na+-K+-2Cl- cotransporter (NKCC2) determines essential functions of the kidney. Results: Annexin A2 (AnxA2) is a new interaction partner of NKCC2 with respect to its adluminal trafficking and activation. Conclusion: Discussion focuses on trafficking, membrane association, and phosphorylation of NKCC2 and its cellular ligand AnxA2. Significance: AnxA2 is relevant for the activation of NKCC2. The furosemide-sensitive Na+-K+-2Cl- cotransporter (NKCC2) is responsible for urine concentration and helps maintain systemic salt homeostasis. Its activity depends on trafficking to, and insertion into, the apical membrane, as well as on phosphorylation of conserved N-terminal serine and threonine residues. Vasopressin (AVP) signaling via PKA and other kinases activates NKCC2. Association of NKCC2 with lipid rafts facilitates its AVP-induced apical translocation and activation at the surface. Lipid raft microdomains typically serve as platforms for membrane proteins to facilitate their interactions with other proteins, but little is known about partners that interact with NKCC2. Yeast two-hybrid screening identified an interaction between NKCC2 and the cytosolic protein, annexin A2 (AnxA2). Annexins mediate lipid raft-dependent trafficking of transmembrane proteins, including the AVP-regulated water channel, aquaporin 2. Here, we demonstrate that AnxA2, which binds to phospholipids in a Ca2+-dependent manner and may organize microdomains, is codistributed with NKCC2 to promote its apical translocation in response to AVP stimulation and low chloride hypotonic stress. NKCC2 and AnxA2 interact in a phosphorylation-dependent manner. Phosphomimetic AnxA2 carrying a mutant phosphoacceptor (AnxA2-Y24DGFP) enhanced surface expression and raft association of NKCC2 by 5-fold upon low chloride hypotonic stimulation, whereas AnxA2-Y24A-GFP and PKC-dependent AnxA2- S26D-GFP did not. As the AnxA2 effect involved only nonphosphorylated NKCC2, it appears to affect NKCC2 trafficking. Overexpression or knockdown experiments further supported the role of AnxA2 in the apical translocation and surface expression of NKCC2. In summary, this study identifies AnxA2 as a lipid raft-associated trafficking factor for NKCC2 and provides mechanistic insight into the regulation of this essential cotransporter. [ABSTRACT FROM AUTHOR]
- Published
- 2014
- Full Text
- View/download PDF
8. Pocket-size hand-held cardiac ultrasound as an adjunct to clinical examination in the hands of medical students and junior doctors.
- Author
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Panoulas VF, Daigeler AL, Malaweera AS, Lota AS, Baskaran D, Rahman S, and Nihoyannopoulos P
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- Adult, Cardiology education, Cardiovascular Diseases diagnosis, Curriculum, Equipment Design, Female, Humans, Male, Medical Staff, Hospital, Physical Examination methods, Prospective Studies, Sampling Studies, Statistics, Nonparametric, Students, Medical, Cardiovascular Diseases diagnostic imaging, Clinical Competence, Echocardiography, Doppler, Color methods, Point-of-Care Systems
- Abstract
Aims: While patient history taking and physical examination remain the cornerstones of patient evaluation in clinical practice, there has been a decline in the accuracy of the latter. Pocket-size hand-held echocardiographic (PHHE) devices have recently been introduced and could potentially improve the diagnostic accuracy of both medical students and junior doctors. The amount of training required to achieve optimal results remains a matter of debate. We hypothesized that the use of PHHE after limited training in the form of a tutorial can improve the clinical diagnosis even in the hands of medical students and inexperienced physicians., Methods and Results: Five final-year medical students and three junior doctors without prior echocardiographic experience participated in a standardized 2 h PHHE bedside tutorial. Subsequently, they assessed 122 cardiology patients using history, physical examination, ECG and PHHE. Their final clinical diagnosis was compared against that of a consultant clinician's and also expert in echocardiography. A total of 122 PHHE were performed of which 64 (53%) by final-year medical students and 58 (47%) by junior doctors. Mean ± SD for diagnostic accuracy after history, physical examination, and ECG interpretation was 0.49 ± 0.22 (maximum = 1), whereas the addition of PHHE increased its value to 0.75 ± 0.28 (Z = -7.761, P<0.001). When assessing left ventricular systolic dysfunction by means of history and physical examination, specificity was 84.9% and sensitivity only 25.9%, whereas after including findings from PHHE, these figures rose to 93.6 and 74.1%, respectively., Conclusion: The use of PHHE after brief bedside training in the form of a tutorial greatly improved the clinical diagnosis of medical students and junior doctors, over and above history, physical examination, and ECG findings.
- Published
- 2013
- Full Text
- View/download PDF
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